The purpose of the project is to study the organization of genes in large DNA molecules obtained from animal viruses. The production and ordering of unique segments of vaccinia virus DNA provide a physical map of the genome. Physical maps have been determined for several different restriction enzymes including SalI, HindIII, XhoI, KpnI, SmaI and BgTI. The last two enzymes are very useful since they cleave the DNA into five easily separated segments from which other restriction enzyme maps can be determined. Experiments designed to lacate the origin(s) of viral DNA replication by electrophoretic analysis of restriction nuclease digests of pulse labeled viral DNA have been initiated. At early times after infection, viral DNA is present in the nucleus and cytoplasm of infected HeLa cells. Early experiments indicate that HindIII segments A, B and C are underlabeled in full size molecules. This result is being examined with other restriction enzymes and conditions. Viral DNA segments are being cloned in plasmids with the aim of doing sequence determination on the region containing the origin of replication and any possible signal elements in the viral genome.